The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D-3.49 aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca2+ concentrations ([Ca-2divided by](j)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca2+](i) and increased potency and efficacy of agonists to elicit Ca2+ responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca2+](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D-3.49 aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations Of alpha(2B)-AR. (C) 2003 Elsevier Science (USA). All rights reserved.